Barrier cream comprising hexamethylenetetramine or derivative thereof

ABSTRACT

The present invention relates to protection agents, in particular to topical compositions such as barrier creams which can prevent the passage of toxic chemicals and chemical warfare agents through the skin. Formulations contain hexamethylene tetramine derivatives or analogues thereof and perfluorinated polymeric compounds.

This application is a 371 of PCT/GB97/01348, filed May 16, 1997.

The present invention relates to skin protection agents, in particularto topical compositions which can prevent the passage of toxic chemicalsthrough the skin, such as barrier creams.

There are many occasions where people are exposed to chemicals or agentswhich have some degree of toxicity and which may penetrate the skin. Itis often desirable or necessary to prevent contact of these chemicals tothe skin as far as possible. For instance, many volatile pesticides canprovide a potential hazard to operators applying them. Examples of suchpesticides include volatile insecticides, particularly organophorphorusinsecticides such as isofenphos, or diethyl toluimide. In otherindustrial applications, workers exposed to industrial solvents such asacetone, chloroform, methanol, hexane, benzene and toluene, may requireprotection.

In chemical warfare situations, highly toxic volatile chemicals, whichmay penetrate the skin can be used. These include Sarin, Soman and Tabunas well as sulphur mustard and Lewisite.

Sulphur mustard (SM) is a vesicating chemical used as a war gas. It is apotent alkylating agent which is thought to be toxic to living tissue byvirtue of its ability to alkylate vital cellular constituents (Fox M.and Scott M., Mut. Res (1980) 75, 131-168). SM has been shown toalkylate DNA, RNA and proteins (Paprimeister B. et al., (1991) MedicalDefence against Mustard Gas (CRC Press) 91-122), though a causal linkbetween alkylation of either of these cellular constituents andvesication has yet to be established.

Although considerable protection from toxic chemical vapours can beafforded by protective clothing in the form of respirators, charcoalcloth garments and gloves these measures may not be completely effectivein all circumstances. The use of a barrier cream might augment thecharcoal cloths used in protective clothing and permit tasks requiringmanual dexterity to be carried out in an atmosphere containing chemicalagent vapour, without gloves for short periods.

Traditional barrier creams have placed a passive barrier between theskin and the environment which prevent access of chemicals generally tothe surface of the skin. Cream bases consist of an emulsion, either oilin water or water in oil (Barry B. W (1983) Drugs Pharmaceutical Sci 18296-350). Water in oil emulsions are unsuitable as barrier creamsbecause the oily deposit they leave on the surface of the skin resultsin loss of tactility. On their own oil in water type creams haverequired layers of greater than 0.56 mm to be established and maintainedon the skin in order to be effective. More recently creams based onsilicone derivatives have allowed effective barriers against generalchemical penetration of the skin to be produced using very thin layersof cream (EP-A-0401840, Japanese Patent Appln no. 57-26610).

Formulation of a reactive chemical which reacts with specific targetchemical groups in the cream converts a passive barrier cream into anactive cream which sequesters and inactivates a specific group of toxicchemicals before they can reach the living layers of the skin.

The use of reactive molecules which would inactivate SM before it couldreact with cell constituents has been investigated. A series ofsulphydryl compounds designed to enter the living cell and fortify itagainst reactive compounds such as SM has already been disclosed in PCTGB91/01462 and GB90148994.5.

It is known that increased levels of GSH and GSH synthetic enzymes(Evans et al., Cancer Res. 47 2525-2530, Ahmed S. et al., J. Biol. Chem.262 15048-15053, and Ahmed S. et al., J. Cellular Physiol. 131, 240-246)confer resistance to nitrogen mustard on cell cultures. In previousstudies attempting to protect cell cultures against SM, Gross C. L. andSmith N. J. (Proc. 1993 Med. Def. Bioscience Rev. (US Army MedicalResearch and Development Command) 1 141-147) found that humanlymphocytes pretreated with 10 mM L-oxothiazolidine-4-carboxylate for 48hours and subsequently exposed to 10 μM SM were 20% more viable than SMonly treated cells 48 hours after exposure. Other attempts to protecthuman lymphocyte cultures (Gross et al., Cell Biology and Toxicology(1993) 9 259-268) by raising intracellular levels of GSH using N-acetylcysteine (10 mM) and subsequently challenging with 10 μM SM increasedviability by only 15 to 19% of control cultures at 48 hours.

The stratum corneum is the dead layer of skin which acts as a barrier tochemical penetration through the skin (Tregear R. T. (1966) TheoreticalExp. Biol. 5, 21-22). The stratum corneum is composed of the dead“protein ghosts” of the living cells of the epidermal layer of the skin,surrounded by a unique mixture of lipids which the cells of theepidermis produce (Wertz P. W. et al., (1989) “Stratum corneum:Biological and Biochemical considerations” in Transdermal Drug Delivery,Ed Hadgraft J & Guy R A. 1-22).

HMT has been used prophylactically to protect rabbits and man againstexposure to lethal phosgene doses (Diller W. F. J.Occ. Med (1978) 20189-193).

The present application relates to a topical composition for useparticularly against SM. In particular the invention relates to anactive barrier cream for SM.

The present invention provides a composition which comprises acomposition for topical application which comprises hexamethylenetriamine or analogues or derivatives thereof.

HMT has the following structure (I)

It is believed that because the structure of HMT contains fournucleophilic nitrogen groups, with a similar electronic structure to N-7in guanine, it neutralises the bifunctional SM more effectively thanconventional monofunctional thiol ligands.

Suitable analogues of HMT are compounds which have organic moietiesconjugated to the HMT cage structure for example of formula (II)

where R¹ is an organic group and X is a anion.

The HMT moiety will hereinafter be represented as #.

Examples of suitable groups for R¹ include optionally substitituedstraight or branched hydrocarbon chains such as alkyl chains, havingfrom 1 to 50 carbon atoms, for example from 6 to 32 carbon atoms. Inparticular, the hydrocarbon chains are optionally substituted by one ormore substituents selected from aryl, such as phenyl, optionallysubstituted by halogen, and/or halogen atoms, such as fluorine, chlorineor bromine and particularly fluorine. A particular group R¹ is afluorobenzyl or a group (CH₂)_(n)CH₃ where n is from 8 to 20, suitablyabout 17.

Further examples of such compounds include compounds of the followingformulae:

(#)—CH₂(CH₂)₂₂CH₃

(#)—CH₂(CH₂)₁₄CH₃

where # represents a hexamethyltetraminyl group.

Suitable anions X are chosen such that the compound of formula (II) arepharmaceutically acceptable salts, in particular those which aresuitable for topical application, for example halide ions such asiodides or bromides.

Suitable derivatives of HMT are made by the conjugation of HMT witheither the normal constituents of barrier creams (such as palmitic orstearic acids), the normal constituents of the stratum corneum(cholesterols or ceramides) or large long chain aliphatic molecules.Suitably the long chain aliphatic molecules have from 16 to 32 carbonatoms. Particular aliphatic molecules which may be employed are fattyacids having from 16 to 32 carbon atoms.

Analogues of formula (II) are suitably prepared by reacting HMT with acompound of formula (III)

Y—R¹  (III)

or a salt thereof; where R¹ is as defined in relation to formula (II)and Y is a leaving group, and thereafter if necessary converting a saltproduced to a different salt.

Suitable leaving groups for Y include halogen such as fluorine, chlorineor bromine, particularly bromine, as well as mesylate and tosylate. Thereaction is suitably effected in an organic solvent, for examplechloroform, alcohols such as methanol or ethanol, acetonitrile atelevated temperatures conveniently at the reflux temperature of thesolvent.

Salts of formula (II) may be any salt as convenient depending upon theparticular nature of the reagents involved. They may subsequently bechanged to a different pharmaceutically acceptable salt using ionexchange procedures, for example by passing the compound down an ionexchange column as is conventional in the art.

Suitably the composition further comprises a cream base.

These compositions place an active barrier between the cellularconstituents of the skin and the outside world, at the level of the deadlayer of the skin, the stratum corneum. This is achieved by a moleculewhich reacts with and sequesters SM which is suitably fixed on thesurface of the strateum corneum in a very thin layer using a barriercream base.

Suitably the composition contains further active reagents, in particularother nucleophilic scavengers. Such groups are useful because SM reactsreadily with a wide range of nucleophilic groups found in biologicalsystems. The most important of these are thought to be the N-7 groups ofguanine residues in nucleic acids and the sulphydryl groups of cysteinecontaining peptides and proteins, the affinity of SM for the formerbiological ligand in particular, dictates the necessity of havingprotective measures in place prior to SM exposure. Since SM isbifunctional it can crosslink biomolecules, forming DNA crosslinksbetween guanine residues (Papirmeister B et al., Medical Defence againstMustard Gas (CRC Press Inc. Bocca) (1991) pp106).

A preferred group of further reagents for use in the composition of theinvention include compounds which have nucleophilic nitrogen and/orsulphur atoms, preferably nitrogen. Examples of sulphur containingnucleophilic compounds include glutathione (GSH) and esters of cysteine,for example as shown in WO 92/04024.

When two different species of nucleophile (ie N and S) are used asextracellular scavengers of SM, they unexpectedly have had an increasedprotective effect in this study, compared to using the compounds singly.Thus a composition which comprises both nucleophilic nitrogen andsulphur are preferred Such compositions include combinations of HMT orderivatives or anlogues and GSH. Alternatively, thiolated forms of theconjugating agents mentioned above, such as thiol derivatives ofcholesterol or a ceramide may be used.

In addition, it has been shown that the use of ligand mixtures increasesthe capacity of protection media to reduce SM-induced cytotoxicitywithout synergistic cytotoxic effects.

Small molecules in a barrier cream can diffuse through the skin and beabsorbed into the blood which may result in local irritation or systemictoxicity. Therefore, in a preferred embodiment, the cream includes largelipophilic molecules which do not penetrate the skin leaving thereactive molecule either on the surface of the skin or retained withinthe superficial layers until they slough off in the normal cycle of theepidermal tissue.

Consequently in a preferred embodiment, the active barrier cream of theinvention is prepared by forming a complex comprising a suitablereactive molecule and a large lipophilic molecule and the resultantcomplex is formulated into a barrier cream base.

A specific embodiment of the invention is a reactive barrier cream whichcomprises HMT in an oil in water emulsion cream base.

However, it has been found that a particularly advantageous carrier forthe compositions comprises a perfluorinated polymeric compound.

Such compounds have a low surface energy and thereby preventpartitioning of the volatile chemical into the topical composition andskin. Indeed it has been found that such compounds can act as effectivebarrier creams when used alone and this forms a further aspect of theinvention.

Suitably the perfluorinated polymeric compound is of formula (IV):

CF₃O—[CF(CF₃)CF₂O]_(n)—[CF₂O]_(m)—CF₃  (IV)

where n and m are independently selected from 4 to 150, suitably from 6to 140.

Suitably the compound of formula (W) is in the form of an oil. Examplesof such compounds are set out in the following Table:

Compound No. n m 1 140  13  2 40 11  3 25 6 4 18 8 5  6 6 6 25 9 7 14 66 16 6

These compounds are available commercially from Aldrich ChemicalCompany, Gillingham, Dorset and are sold under the trade name“Fomblin”™. Other compounds of formula (IV) may be prepared usingconventional methods.

The perfluorinated compounds produce a significantly improved barrier toSM than conventional oil-in-water emulsions, which under someconditions, may actually trap the SM and increase the penetration rate.

The compositions of the invention comprise a substantial portion ofperfluorinated polymeric compound for instance up to 100% w/wperfluorinated compound. Suitably, the compositions comprise from30-100% w/w perfluorinated compound and preferably in excess of 50% w/w.Where 100% prefluorinated compound is used, this must comprise an oil.

These compositions may further comprise active agents other than or inaddition to HMT or analogues or derivatives thereof as described above,which react with or sequester the chemical which it is intended shouldbe prevented from reaching the skin.

For instance, reactive molecules which would inactivate for example, SMbefore it could react with cell constituents may be included. Suchcompounds include compounds disclosed in PCT GB91/01462 and GB90148994.5or WO 92/04024 as mentioned above. A particular reactive agent which maybe used in the formulations of the invention is potassium butadionemonoximate.

Where present, preferred ratios of the active compound: perfluorinatedcompound in a barrier cream formulation are, as before, from 5:95 to60:40w/w for instance from 10:90 to 60:40 w/w, most preferably 25:75w/w.

The compositions may be in the form of oil in water or water in oilemulsions. Other components of such emulsions include emulsifying agentsand oils conventionally used in barrier cream type formulations such asmineral or silicone oils. However, the compositions are preferably oilsrather than emulsions.

Preferably, the compositions of the invention finder contain lowermolecular weight perfluorinated compounds such aspolytetrafluoroethylene. These compounds can affect the viscosity of theformulation, making them more effective barriers. Suitable compounds ofthis type are available from ICI plc. They have a molecular weight ofthe order of 10⁶, and a particle size of approximately 6 microns.

The present inventors have studied the ability of HMT to protect cellsagainst the toxic effects of SM in culture and to prevent thepenetration of SM through isolated skin, to assess the feasibility ofits prophylactic use to prevent SM induced skin injury. Penetration ofradiolabelled SM across human stratum corneum has been measured in vitrousing glass diffusion cells.

In the cell culture study, Simian virus keratinocyte-14 (SVK-14—a humankeratinocyte line transformed with the SV40 virus) cell cultures wereused to assess the protection afforded by a prophylactic mixture ofsulphur and nitrogen containing compounds against SM, and the effect ofusing the compounds singly. Mitotically active cells are known to bemore sensitive to the effects of DNA alkylating agents such as SM (FoxM. & Scott M. et al., Mut. Res. 75 131-168), since the formation ofcross-linked DNA lesions prevents mitosis, leading to unbalancedmetabolism and cell death (Cohen et al., Proc. NY Nat. Acad. Sci. 40885-893).

The use of an actively mitosing, keratinocyte-derived cell line as usedin the present study has several important advantages over restingperipheral blood lymphocytes used in the studies of Gross et al.(supra): firstly, the epithelial morphology of the SVK culture meansthat it is a better model of the main targets of SM intoxication, namelythe basal epidermis, the broncho-epithelium and the corneal epithelium;secondly, SVKs are an attached cell-line, enabling the study of specificintercellular interactions following SM exposure; finally, because theSVK cultures are mitotically active they will be more susceptible to SMfor the reasons outlined above.

Altogether, the use of SVK cultures represents a more severe test of theefficacy of putative prophylactic regimes than does the use ofperipheral blood lymphocytes. Consequently, the level of protection ofthe SVK cultures observed in this study is far greater than thatachieved in previous studies aimed at increasing intracellular GSHlevels.

In the present tests, assessment of the pretreated cultures 96 hoursafter exposure to SM demonstrated a significant increase in theviability of the SVK cells compared to those exposed to SM. Cultureviability was measured using the NR assay. This involves the uptake ofNR dye into intracellular vesicles such as lysosomes and gives a measureof cellular activity as well as membrane integrity.

63.3% of cells in cultures pretreated with 4 mM GSH/10 mM HMT remainedviable compared to 33.5% of SM exposed cells, this was mirrored in thephotomicrographs (not included) showing SM exposed and pretreatedcultures respectively. SM exposed cells have taken up the dye but thedrastically altered cellular morphology (extended cytoplasm andcongregation of lysosomes around the swollen nucleus) indicates thatthese cells are not in a truly viable and proliferative state. Incomparison, the pretreated cultures show protection of the cells with apreserved monolayer, a great reduction in the number of hyperplasmiccells and a more diffuse distribution of the lysosomes in the cytoplasm.

Cell numbers were measured using crystal violet staining of DNA.Pretreatment of the SVK cultures with 4 mM GSH/10 mM HMT did notsignificantly alter the growth rate compared to the control, however thedifference between pretreated cultures compared to those exposed to SMis marked. Not only is there a preservation of cell numbers at everytime-point but also the rate of culture depopulation is less and thereis an eventual recovery of the population by 96 hours at a comparablegrowth rate to the control. The use of either 8 mM GSH or 10 mM HMTsingly was not as effective as using the compounds in combination at the72 hour time-point, with increases in viability of 19.8% or 22.6%compared to 29.8%. The use of 16 mM GSH gave an increase in viability of20.6% at 72 hours compared to the SM control (p<0.001), whilst the useof 20 mM HMT did not alter the toxicity of SM (p>0.05) at 72 hours. Theuse of GSH and HMT in combination confered greater protection on thecultures than higher concentrations of the single prophylactics.

Penetration of radiolabelled SM across rubber latex membranes and humanepidermal skin membranes has been measured in vitro using glassdiffusion cells is also reported hereinafter.

In a diffusion cell system the chemical diffuses through the membrane(the penetrant) by partitioning between its vehicle and the membrane,then between the membrane and the receptor fluid. When the thermodynamicactivity, of the penetrant (usually synonymous with concentration) onboth sides of the membrane is equal the system is in equilibrium andthere will be no further change in the amount of penetrant in thereceptor fluid.

In certain diffusion studies reported hereinafter, the ability of eachcomposition to slow the rate of penetration through the membrane wasshown by a calculation of the retardant index (RI) using the equation

RI=J _(max)(control)/J _(max)(treated)

where J_(max) is the maximum penetration rate of sulphur mustard throughcontrol (untreated) and treated membranes.

When a compound reacts with the penetrant on the surface of the membraneand forms a product which does not penetrate the membrane, the totalamount of penetrant in the receptor fluid at equilibrium will bereduced. In such a case, and if the penetrant is applied as an undilutedliquid the measured maximum rate of penetration would only be reduced ifthe presence of the product reduces the thermodynamic activity of thepenetrant.

When using radiolabel to measure the amount of penetrant in the receptorchamber no distinction can be made between unreacted penetrant andproduct. Therefore if the product also penetrates the membrane themeasured maximum diffusion rate and total amount penetrating will beincreased.

When the reactive protectant is applied to the membrane in a cream basethe penetrant must partition into the cream and then into the membranein order to enter the receptor chamber. The partitioning of thepenetrant into the cream can thus modify the rate of penetration byaltering the partitioning of the penetrant into the membrane. Thereactive protectant must also retain its reactivity with the penetrantwithin the chemical environment of the cream base.

In diffusion studies reported hereinafter, the reduction in the totalamount of SM penetrating the epidermal membranes pretreated with HMTalone, without effect on the maximum flux rate, supports the hypothesisthat HMT is reacting with SM to form a product which does not cross themembrane.

Mixing HMT with oil in water cream bases retains the reduction in totalamount penetrated indicating that any reaction also occurs in thismedium. The reduction in maximum flux rate observed in a cream base,such as Stokoderm® obtainable from Arco Skin Care Products, Hull, mayresult from a reduction in partitioning of SM into the epidermalmembranes from the cream. The possibility that the product of anyreaction may partition into the membrane from the cream base cannot bediscounted and may explain the slight increase in maximum penetrationrate measured through the oil in water emulsions containing beeswax andbrij 52.

In summary, the reduction in the cytotoxicity of SM and its penetrationof epidermal membranes in the presence of HMT support the use of HMT orone of its analogues as a reactive constituent of barrier creams.

As illustrated hereinafter, formulations referred to in this applicationcan reduce the rate of penetration of SM through isolated stratumcorneum by five fold, and sometimes up to 45 fold, and reduce the totalamount penetrated by up to 90%.

BRIEF DESCRIPTION OF DRAWINGS

The following Examples illustrate the invention. In these examplesreference is made to the attached Figures in which:

FIG. 1 is a graph showing the effect of 2 (▾), 10 (▪), 50 (▴) and 250 μMSM () on the viability (Neutral Red assay) of SVK-14 cultures withtime. Data are shown as the mean±S.E.M. (n=8) expressed as a percentageof the untreated control;

FIG. 2 shows the effect on viability of pretreatment of SVK-14 cultureswith a mixture of 4 mM GSH/10 mM HMT prior to exposure to 10 μM SM.Viability of pretreated cultures (▾) and those only exposed to SM (▴)was measured by the NR assay. Data are shown as the mean±S.E.M. (n=15)expressed as a percentage of the untreated control;

FIG. 3 shows the effect on cell numbers of pretreatment of SVK-14cultures with a mixture of 4 mM GSH/10 mM HMT prior to exposure to 10 μMSM. Cell numbers of untreated control cultures (Δ), pretreated controlcultures (∇), pretreated cultures exposed to SM (▾) and untreatedcultures exposed to SM (▴) was measured using the crystal violet assay.Data are shown as the mean±S.E.M.(n=15);

FIG. 4 is a graph showing penetration of ³⁵S-radiolabelled sulphurmustard through human epidermal membranes. Points are mean±SD (n=4 to6); where represents control, no barrier cream; represents HMT alone, isHMT in Stokoderm base (20:80w/w) and ⋄ is HMT is Stokoderm basecontaining PTFE (20:50:30 w/w);

FIG. 5 is a graph showing penetration of ³⁵S-radiolabelled sulphurmustard through human epidermal membranes. Points are mean±SD (n=4 to6). HMT was mixed with each of the following cream bases at 30% (w/w)where represents control, no barrier cream; represents 50% water, 5%beeswax, 15% Brij 52 and 30% heavy white mineral oil (% w/w), is 50%water 5% beeswax, 15% Brij 52 and polymethylhydrosiloxane (% w/w) and ▪is 50% water, 5% beeswax, 15% Brij 52 and 30% Dow Corning fluorinatedoils (Fs300:Fs10000, 1:1)(% w/w); Polycysteine with a chain length of5-15 kD was formulated into the following cream base at 30% -⋄-50% water5% beeswax, 15% Brij 52 and heavy white mineral oil (% w/w);

FIG. 6 is a graph showing the ability of perfluorinated polymers toretard the rate of penetration of SM (expressed as retardation index).“Stoko” is an abbreviation for the commercially available barrier creamStokoderm® consisting of an oil-in-water emulsion and was included forcomparison (positive control);

FIG. 7 is a graph showing the amount of SM penetrated through pretreatedmembranes expressed as percentage of control amount penetrated after 3hours;

FIG. 8 is a graph showing SM penetration through human skin underunoccluded conditions pretreated with Fomblin-Z-diol™ (), a comerciallyavialable barrier cream (Stokoderm™) with a reactive thiol ester (▴) andan untreated control (♦);

FIG. 9 is a graph showing SM penetration under unoccluded conditionsthrough skin pretreated with a formulation in accordance with theinvention (▴) and an untreated control (♦);

FIG. 10 shows the percentage radiolabelled SM distribution in a receptorchamber (penetrated SM), skin, donor chamber (reacted SM) and vapour;and

FIG. 11 shows an autoradiograph of a TLC plate from samples of HMT andanalogues which had been allowed to react with radiolabelled SM.

EXAMPLE 1 Cell Culture Experiments

A human keratinocyte culture, specifically SVK-14 human keratinocytecells were used in these experiments. Tissue culture plastics wereobtained from J. Bibby Science Products Ltd, Stone, Staffs. The SVKcells were grown in a medium comprising 84% v/v Dulbecco's modifiedEagle's medium (DMEM), 16% v/v foetal calf serum (FCS), containing 100IU/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine (referred tohereinafter as SVK medium). The cultures were maintained in a 37° C.incubator in a humidified atmosphere of 95:5 air:carbon dioxide. Thecells were grown to confluency in 150 cm² flasks, removed from theflasks by incubation at 37° C. with a trypsin/EDTA solution (Sigma), for5-10 minutes, sown in 96 well cell culture plates (40,000 to 60,000cells per well) and allowed to attach for 24 hours.

Solutions of GSH (8 mM and 16 mM) and HMT (10 mM and 20 mM) in DMEM,containing 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mMglutamine, were mixed to give four solutions herein after referred to asprotection media:

1 4 mM GSH, 5 mM HMT; 2 4 mM GSH, 10 mM HMT; 3 8 mM GSH, 5 mM HMT; 4 8mM GSH, 10 mM HMT,

Protection media and single solutions of 8 mM GSH and 10 mM HMT,prepared as above, were sterile filtered before use.

The response of the SVK-14 cell line to sulphur mustard exposure wasfirst assessed by comparing dose response curves for four concentrationsof SM (2, 10, 50 and 250 μM) at time points up to 72 hours (FIG. 1).Cell viability was assayed using neutral red (NR) dye retention(Griffiths et al, Toxicology (1994) 90 11-27) at various time pointsfollowing SM exposure. Results are expressed as a percentage of livecontrol (non-SM exposed) cultures.

The deleterious effects of SM on cell viability were found to be bothtime and concentration dependant and the time taken to reach the LC₅₀(Lethal concentration 50-the concentration which reduces cell cultureviability to 50% of control levels) was inversely proportional to theconcentration of SM used (2, 10, 50 and 250 μM SM took 47, 35, 15 and 8hours respectively). Subsequently 10 μM SM was used as a standard doseagainst which the efficacy of the pretreatment could be assessed.

Ninety six well culture plates were pretreated by replacing the culturemedium for 100 μl of one of the protection media or 100 μL DMEMcontaining, 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mMglutamine, as non-pretreated controls. The cultures were then incubatedin the presence of these solutions for 37° C. in a humidified atmosphereof 95:5 air:carbon dioxide. After 1 hour 100 μl SM solution in Hank'sbuffered salt solution (HBSS) (10 μM, prepared by dilution of a 50 mMstock solution in isopropanol) was added to each well and the platesincubated for a further 1 hour at 37° C. in the same atmosphere. Thesolutions were removed from the wells and 100 μl of SVK medium wasadded. The cultures were incubated at 37° C. in the atmosphere describedabove for periods of up to 96 hours. At selected time points, viabilitywas determined by the NR and crystal violet assay.

It was found that the addition of SM (10 μM) to the SVK culturesresulted in a steady decline in viability over time so that at 24, 48,72 and 96 hours viability (as determined by the NR assay, FIG. 2) hadfallen to 85.7, 54, 40.8 and 33.5% of the control cultures. Thiscontrasts with cultures to which SM was applied in the presence of 4 mMGSH/10 mM HMT, for viability was then maintained at 99, 69.7, 58.3 and63.3% respectively at the above time points. The viability of thepretreated cultures was therefore 1.9-fold (or 89%) greater thannon-protected cultures 96 hours after SM exposure. This difference wassignificantly different (p<0.001) when measured by ANOVA with Tukey posttest.

Cell number was determined by nuclear staining with 0.1% (w/v) crystalviolet after the method of Gillies et al, (Analytical Biochem. (1986)159 109-113). Results were expressed as the optical density measured at620 mn.

The results are shown in FIG. 3 and are broadly similar, although at 96hours the number of cells in the pretreated cultures was 2.3-fold (or131%) greater than in the unprotected cultures. This difference was alsosignificantly different (p<0.001) when measured by ANOVA with Tukey posttest.

The other protection media comprising different combinations of GSH/HMTprotectants conferred similar levels of protection on SVK cell culturesexposed to SM when measured by NR assay (Table 1) and crystal violetassay (Table 2).

TABLE 1 Relative viability of pretreated cultures compared to culturesonly exposed to 10 μM SM {Private} Drug Combination 24 h 48 h 72 h 96 h10 μM SM  93.8 ± 0.56 59.1 ± 0.48 40.0 ± 0.57 28.7 ± 0.28 4 mM GSH/100.3 ± 0.56* 74.3 ± 0.48* 50.9 ± 0.85* 55.1 ± 0.55* 5 mM HMT + 10 μM SM10 μM SM  85.7 ± 1.4 54.0 ± 0.52 40.8 ± 0.83 33.5 ± 0.27 4 mM GSH/  99.0± 1.4* 69.7 ± 1.82* 58.3 ± 0.56* 63.3 ± 0.53* 10 mM HMT + 10 μM SM 10 μMSM  93.4 ± 0.57 63.2 ± 0.75 38.2 ± 0.9 31.0 ± 0.54 8 mM GSH/ 101.1 ±0.85* 80.7 ± 0.5* 63.9 ± 0.3* 64.2 ± 0.54* 5 mM HMT + 10 μM SM 10 μM SM101.9 ± 2.3 70.2 ± 0.72 35.3 ± 0.91 26.9 ± 0.55 8 mM GSH/ 116.4 ± 2.3*84.9 ± 0.96* 65.0 ± 0.96* 62.4 ± 1.1* 10 mM HMT + 10 μM SM Mean data areshown with standard error of the mean for groups of 15 cultures (as apercentage of the control mean). * shows significant difference (p <0.001) when measured by ANOVA with Tukey post test.

TABLE 2 Comparison of cell number between pretreated cultures and thoseonly exposed to 10 mM SM Drug Combination 24 h 48 h 72 h 96 h Control0.161 ± 0.179 ± 0.218 ± 0.249 ± 0.001 0.001 0.002 0.001 10 μM SM 0.132 ±0.069 ± 0.056 ± 0.052 ± 0.002 0.001 0.001 0.001 4 mM GSH/5 mM HMT +0.134 ± 0.101 ± 0.099 ± 0.103 ± 10 μM SM 0.003 0.001* 0.001* 0.001*Control 0.178 ± 0.200 ± 0.223 ± 0.244 ± 0.002 0.002 0.002 0.002 10 μM SM0.141 ± 0.074 ± 0.049 ± 0.052 ± 0.002 0.001 0.001 0.002 4 mM GSH/10 mMHMT + 0.162 ± 0.110 ± 0.094 ± 0.119 ± 10 μM SM 0.002* 0.002* 0.002*0.001* Control 0.151 ± 0.162 ± 0.186 ± 0.228 ± 0.002 0.004 0.003 0.00210 μM SM 0.123 ± 0.057 ± 0.032 ± 0.045 ± 0.001 0.002 0.002 0.001 8 mMGSH/5 mM HMT + 0.138 ± 0.080 ± 0.085 ± 0.115 ± 10 μM SM 0.002* 0.002*0.002* 0.002* Control 0.147 ± 0.175 ± 0.202 ± 0.246 ± 0.001 0.002 0.0010.001 10 μM SM 0.125 ± 0.067 ± 0.047 ± 0.042 ± 0.001 0.001 0.001 0.001 8mM GSH/10 mM HMT + 0.136 ± 0.110 ± 0.096 ± 0.104 ± 10 μM SM 0.002*0.002* 0.002* 0.002* Data are shown as the mean absorbance (620 nm) withstandard error of the mean for groups of 15 cultures. * showssignificant difference (p < 0.001) between SM only and treatment + SMwhen measured by ANOVA with Tukey post test.

Pretreatment with 8 mM GSH only resulted in 111.2, 91 and 60.8%viability when measured by NR assay at 24, 48 and 72 h post exposurerespectively (96 h not assayed), compared to the SM-only exposed cellswhich were 102.9, 68 and 41% viable at the same time points.Pretreatment with 16 mM GSH only resulted in 102.5, 86.8, 76.2 and 80.7%viability when measured by NR assay at 24, 48, 72 and 96 hours postexposure respectively, compared to the SM-only exposed cells which were97.2, 74.5, 55.6 and 49.1% viable at the same time points. Pretreatmentwith 10 mM HMT only resulted in 88.9, 83.5 and 59.4% viability whenmeasured by NR assay at 24, 48 and 72 hours post exposure respectively(96 hours not assayed), compared to the SM-only exposed cells which were91.7, 71.4 and 42.2% viable at the same time points. Pretreatment with20 mM HMT only resulted in 93.9, 97, 89.2 and 72.2% viability whenmeasured by NR assay at 24, 48, 72 and 96 h post exposure respectively.Treatment with 20 mM HMT and 10 μM SM resulted in 96.8, 72.3, 41.6 and12.7% viability when measured by NR assay at 24, 48, 72 and 96 hourspost exposure respectively, compared to the SM-only exposed cells whichwere 99.2, 63.0, 41.8 and 30% viable at the same time points.

When stained with crystal violet, control cultures and those pretreatedwith protection media demonstrated the epithelial cobblestone morphologyand the small cytoplasmic/nuclear ratio normally seen in a confluentmonolayer of SVK cells. The application of 10 μM SM to these culturesresulted in:

1) an enlarged cytoplasmic/nuclear ratio and nuclear swelling

2) plasma membrane blebbing leading to the formation of cell fragments

3) necrotic bodies

4) loss of cell-cell contact and normal epithelial morphology

5) an overall reduction in cell numbers

Staining of SM exposed cells with NR showed an accumulation of lysosomesaround the nucleus in addition to the above features. The observedchanges were time dependant, the longer time points (48-96 hours) showeda progressive destruction of the SVK cultures.

Treatment of SVK cultures with any of the combinations of protectionmedia prior to SM exposure resulted in cultures which after crystalviolet staining demonstrated:

1) only limited cytoplasmic swelling

2) preserved epithelial morphology

3) fewer necrotic bodies

4) evidence of mitotic activity

In addition, neutral red staining showed a more diffuse distribution ofthe lysosomes in the cells. These features were observed 96 hourspost-exposure.

Treatment of SVK cell cultures with GSH, HMT or combinations of GSH andHMT did not significantly alter the growth of the cells over the courseof the experiment when measured by the crystal violet assay, nor altercellular function when measured by neutral red uptake, indicating thatthese compounds and combinations were biocompatible.

EXAMPLE 2 Topical Skin Protectant Formulations

Topical skin protectants were formulated using a mixture of barriercream comprising stearic acid sold under the trade name Stokoderm®,protective compound and optionally with polytetrafluoroethylene, (PTFE).The composition of the formulations is given in Table 4 hereinafter.

Initially, the formulations were produced by freezing the componentcompounds in liquid nitrogen with subsequent mixing in a grinding mill(A10 model, IKA Labortechnik, Neumagenstraβe, Germany) for a total oftwo minutes.

A more complete mixing of the components was found to be achieved by theuse of a low volume stirrer. This apparatus consists of a stainlesssteel tube, closed at one end with a screw-in base and a router bit witha rubber seal connected to a variable speed drill. The compounds wereplaced into the tube and mixed with the router bit. A 100% recovery ofcream was achieved after mixing by unscrewing the base and inserting theplunger of a 10 ml syringe

EXAMPLE 3 Diffusion Studies

Epidermal membranes consisting of predominantly stratum corneum withsome attached keratinocytes was obtained from human skin taken duringabdominal reduction operations and free of overt pathology. The skin wasfrozen and stored at −20° C. after excision until required. Theepidermis was isolated by thawing the skin, removing the underlyingfatty tissue and immersing in water at 60° C. for 45 seconds. Theepidermal membranes could then be scraped away using a pair of forceps,floated on distilled water and mounted on aluminium foil dried in theair until excess water had evaporated then frozen and stored at −20° C.until required.

Assessment of topical skin protectants was carried out using Franz-typeglass diffusion cells as previously described (Jenner et al., J. Pharm.Pharmacol. (1995) 47 206-212), with an area available for diffusion of2.54 cm². Each diffusion cell consisted of an epidermal membrane placedon a metal gauze support forming a barrier between an upper (donor) andlower (receptor) chamber, both of which were initially filled with 0.9%saline.

The structural integrity of each membrane was determined by electricalresistance as previously described (Chilcott R. P. et al, Human andExperimental Toxicology, 14 (9) p773). Membranes with a resistance ofless than 3.0 kΩ were judged to be damaged and were rejected from thestudy.

Following electrical resistance measurement, the receptor chamber fluidwas replaced with 50:50 aqueous ethanol and the saline in the donorchamber was removed. Each formulation was applied as a 200 μl volume,giving a nominal thickness of 0.78 mm on the epidermal membrane surface.The protective compounds were also assessed individually withoutformulation by adding 200 mg directly to the donor chamber. Eachformulation or compound was applied two hours before the addition of 20μl [³⁵S]SM (50 μCi.ml⁻¹) as an airbourne droplet to the donor chamber.The experiments were subsequently conducted under occluded conditions inthat the donor chamber was sealed with a plastic lid using contactadhesive (Bostik All Purpose Clear Adhesive).

Samples (20 μl) of receptor chamber fluid were taken at regularintervals for up to 60 hours. Each sample was replaced with anequivalent volume of 50:50 aqueous ethanol. The amount of radioactivityin each sample was measured by liquid scintillation counting in 5 mlEmulsifier Safe scintillation cocktail (Canbera Packard, Michigan USA),using a Rackbeta II 1215 scintillation counter. Amounts of radioactivitywere related to amounts of SM determined from a sample of donor chamberliquid taken at the start of the study.

Results were analysed using a spreadsheet program (Excel, MicrosoftCorp.) on a Mitac 486DX microcomputer. Results are presented as graphsof amount of radiolabelled SM penetrated with time and are shown inFIGS. 4 to 5. Where applicable, the maximum penetration rate achieved(Jmax), amounts penetrated at 6, 12 and 24 hours and total amountpenetrated are summarised in Tables 2 and 3.

When used to prevent SM penetration through isolated epidermal membranesHMT, alone or formulated in Stokoderm cream reduced the total amount ofSM which penetrated over 48 hours (FIG. 4).

HMT alone reduced the total penetrated to 42%±14% of non-pretreatedcontrols, whereas when mixed with an oil in water cream base (Stokoderm)17%±3% of controls penetrated. The addition of PTFE to the HMT:Stokodermmixture allowed more SM to penetrate than HMT:Stokoderm, but not as muchas HMT alone, 27%±10% of non-pretreated controls (Table 3).

TABLE 3 Summary of Percentage of Average Control Amount PenetratedFollowing Pretreatment With Topical Skin Protectants % CONTROL AMOUNTPENETRATED PRETREATMENT 6 h 12 h 24 h 48 h HMT 116 ± 124 103 ± 64 60 ±28 42 ± 14 HMT in Stokoderm base 18 ± 5  19 ± 4 19 ± 4  17 ± 3  (20:80,w/w) HMT in stokoderm base with 61 ± 26  71 ± 30 46 ± 12 27 ± 10 PTFE(20:50:30, w/w) % Control amount penetrated = the amount of SMpenetrated through pretreated in comparison to control membranesexpressed as a percentage at 6, 12 and 24 hours and at the terminationof the experiment.

HMT alone had no effect on the maximum flux of SM penetration but mixingHMT with Stokoderm base or Stokoderm base containing PTFE resulted inmaximum fluxes of 48±20 μg·cm⁻²·hr⁻¹ and 178±86 μg·cm⁻²·hr⁻¹respectively (non-pretreated controls=233±181 μg·cm⁻²·hr⁻¹) (Table 4).

TABLE 4 Quantitative Summary of SM Penetration Rates FollowingPretreatment With Topical Skin Protectants Jmax TREATMENT (μg.cm⁻².hr⁻¹)control (n) 233 ± 181 HMT in Stokoderm base (20:80, w/w)(n) 48 ± 20 HMTin stokoderm base with PTFE (20:50:30, w/w)(n) 173 ± 86  HMT (n) 345 ±278 Number in brackets indicates the number of epidermal membranes usedin each study.

Formulation of HMT into an oil in water cream base consisting of water,beeswax, brij 52 and one of three oils (heavy white mineral oil,polymethylhydrosiloxane or fluorinated oils) also produced creams whichreduced the total amount of SM penetrating over 48 hours but did notreduce the maximum flux rate (FIG. 5).

EXAMPLE 4 Diffusion Studies Using Perfluorinated Polymer Barrier Creams

Assessment of topical skin protectants was carried out using Franz-typeglass diffusion cells substantially as previously described (Jenner etal., J. Pharm. Pharmacol. (1995) 47 206-212), with an area available fordiffusion of 2.54 cm². In this case however, each diffusion cellconsisted of a rubber latex membrane (26% ethylene vinyl acetate (EVA),RMCS Shrivenham) placed on a metal gauze support forming a barrierbetween an upper (donor) and lower (receptor) chamber.

Previous studies had shown that this apparatus, including the rubberlatex membrane would provide a suitable model for identifying compoundswith a barrier function and that penetration rates provide aquantitative method of assessing the retardant properties of thecompounds.

The receptor chamber fluid was filled with 50:50 aqueous ethanol. Eachcomposition was applied as a 200 μl volume, giving a nominal thicknessof 0.8 mm on the membrane surface. Each composition was applied twohours before the addition of 20 μl [³⁵S]SM (50 μCi·ml⁻¹) as an airbornedroplet to the donor chamber. The experiments were subsequentlyconducted under occluded conditions in that the donor chamber was sealedwith a plastic lid using contact adhesive (Bostik All Purpose ClearAdhesive).

Samples (20 μl) of receptor chamber fluid were taken at regularintervals for up to 8 hours. Each sample was replaced with an equivalentvolume of 50:50 aqueous ethanol. The amount of radioactivity in eachsample was measured by liquid scintillation counting in 5 ml EmulsifierSafe scintillation cocktail (Canbera Packard, Michigan USA), using aRackbeta II 1215 scintillation counter. Amounts of radioactivity wererelated to amounts of SM determined from a sample of donor chamberliquid taken at the start of the study.

Results were initially plotted as total amount of ³-S-radiolabelledsulphur mustard penetrated against time. During the first four hours, astraight line is observed (the maximum penetration rate, J_(max)). Fromthis, the retardant index could be calculated and the results are shownin FIG. 6

In addition, the total amount of SM penetrated through the membranesover three hours was calculated and expressed as a percentage of theuntreated control. The results are shown in FIG. 2.

It can be seen from these Figures that the compositions of the inventionprovide significant protective barrier effects and are more effectivethan a commercially available barrier cream.

EXAMPLE 5 Comparison of SM Penetration through Skin Pretreated withPerfluorinated Polymer Barrier Oil and Commercially Available BarrierCream

Using methodology substantially as described in Example 3 above, theeffect of pretreating the skin samples with Fomblin-z-diol™ two hoursbefore addition of liquid SM is shown in FIG. 8. The experiment waseffected under unoccluded conditions (where the SM is free to vaporiseoff the skin surface), and a standard barrier cream (an oil-in-wateremulsion) containing a reactive thiol compound and an untreated controlwas used for comparison purposes.

It was found that under these conditions, the oil-in-water creamincreased the total amount of mustard penetrated. In practical terms, auser “protected” with this cream would receive a higher dose andpossibly a more severe mustard skin injury than unprotected bare skin.In contrast, the Fomblin provided protection for up to 35 hours. Thisprotection is significant for the first 12 hours. Furthermore, thedroplet of mustard on the Fomblin treated skin surface can be easilymoved, indicating that on a vertical surface, very little or no mustardwould penetrate. The maximum protective effect of Fomblin-z-diol(measured as the percentage decrease in total mustard penetrated) wascalculated as 92% at 3 hours, 84% at 12 hours and 61% at 24 hours.

EXAMPLE 6 Effect of Mixture of Fomblin and PTFE

A formulation was produced by mixing 76% Fomblin HC/R™ and 24% PTFE(polytetrafluoroethylene) at 1800 rpm at room temperature for 4 hours.Example 5 was then repeated using this formulation and an untreatedcontrol. The results are shown in FIG. 8.

It is clear that the inclusion of PTFE has had a profound effect in thatthe pretreated skin has approximately 75% less mustard penetrated thancontrols, and this decrease is present for the whole experiment (over 45hours). The maximum protective effect of this formulation (measured asthe percentage decrease in total SM penetrated) was 90% at 3 hours, 90%at 12 hours, 75% at 24 hours and 75% at the end of the experiment.

This was found to be better than the use of Fomblin HC/R alone which isindicative of the beneficial effects of PTFE.

EXAMPLE 7 Formulation of Active Barrier Creams

The following formulations were prepared:

Formulation A:

93% Fomblin™ HC/R

6.6% aqueous saturated HMT solution

0.4% Brij 30

0.4% cetyl alcohol

A small volume of Fomblin™ HC/R was mixed with the Brij 30 and cetylalcohol at 50° C. and the mixture added to the rest of the Fomblin™ HC/Rand saturated HMT solution at room temperature with constant mixingusing a Hiedolf constant torque stirrer at 1800 rpm.

Formulation B

0.08% Brij 52

1.16% aqueous saturated HMT

98.76% Fomblin™ HC/R

In this case, Fomblin™ HC/R was heated to 40° C. and the Brij 52 and HMTsolutions were added. The mixture was heated to 70° C. with constantstirring (1800 rpm) and allowed to cool to room temperature also withconstant stirring.

The formulations were subjected to diffusion studies as descibed abovein Example 3 under unoccluded conditions. The reactivity of the barriercreams was qualitatively measured by measuring the amount ofradioactivity remaining in the donor chamber as compared to thereceptor, the vapour and the skin at the end of the experiment, when nomore penetration of radiolabelled SM was occuring. The results are shownin FIG. 10.

The control (un-pretreated) skin had little or no mustard on the surface(donor chamber) whereas both reactive barrier cream formulationscontained radioactivity, implyin that the radiolabelled mustard hadreacted with the HMT to form a stable, non-penetrating adduct. This wasfurther substantiated be the fact that similar formulations without HMThad residual donor chamber radioactivity equal to that of the control.

EXAMPLE 8 Preparation of N-octadecyl Hexaminium Halide Salts

1-bromo octadecance (12.0 g) was added to sodium iodide (5.5 g) inmethyl iso butyl ketone and the stirred suspension refluxed for 3 hours.The suspension acquired an opalescent appearence after 25 minutes.Estimation of conversion was made by determining unreacted iodide withcopper sulphate. A conversion of approximately 90% was achieved and thismaterial was used without further purification.

I-iodo octadecane (crude, 13.3 g) was refluxed with hexamethylenetetramine in chloroform (40 ml) as previously. The suspension clearedbriefly and then a precipitate formed. After cooling and stirring for 40hours, the white solid was filtered and washed with chloroform (2×7 ml)and then cyclohexane (7 ml) and then dried in vacuo at 30-40° C.

Yield: 3.9 g.

The ionic iodide was determined argenotmetrically: 24.03%; (theoretical24.37%), i.e. 98.6% of the expected figure. Melting point: 146-150° C.with decomposition. The structure was confirmed by IR.

This product may be converted to the bromide salt using a suitable ionexchange column.

EXAMPLE 9 Preparation of 2 Pentafluorobenzyl Hexaminium Bromide

In dry glassware, α-bromo-2,3,4,5,6-pentafluorotoluene (5.02 g) andhexamethylene tetramine (3.0 g) were refluxed in chloroform (20 ml). Theheadspace was purged in nitrogen and a drying tube was used to maintainanhydrous conditions. The suspension was warmed to effect solution. Aprecipitate formed after a few minuted and thickened over 40 minutes. Atemperature rise of 3 degrees above ambient was experienced. Afterstirring for 2.5 hours at ambient temperature, the suspension was takento reflux and allowed to cool. The thick paste was filtered and washedwith chloroform (3×5 ml). The resulting solid was dried in vacuo at30-40° C. Yield: 6.84 g. m.p. 206-208° C., decomposition. Watersolubility: 1 g dissolves in 11 ml at 20° C. (equivalent to 90 mg·ml⁻¹).

The ionic bromide was determined using an excess of silver nitratesolution followed by back-titration with potassium thiocyanate to givean assay of 99.3%. The structure of the compound was confirmed by I.R.

EXAMPLE 10 Reactivity of HMT Analogues

The reactivity of the analogues of HMT produced in Examples of 8 and 9above was tested as follows. The following solutions were prepared:

a. HMT (140.19 mg) in THF (10 ml).

b. N-octadecyl HMT (406.7 mg) in THF (10 ml)

c. Perfluorobenzyl HMT (387.46 mg) in tetrahydrofuran (THF) (10 ml);

To each solution, radiolabelled sulphur mustard (124.9 μl) (³²S) wasadded. After one hour, 20 μl samples were removed and placed onto asilica TLC plate in lanes 2-4 respectively with a drop of ³⁵S-SM(lane 1) and were chromatographed with a THF mobile phase forapproximately 40 minutes. The TLC plate was then removed andautoradiographed using Kodak Biomax x-ray film.

The TLC results are shown in FIG. 11. It is noticeable that free H isonly present in the contol sample. No free H is present in solutionscontaining reactive compounds HMT and the octadecyl and perfluorobenzylanalogue thereof.

What is claimed is:
 1. A barrier cream composition comprising 90-99% w/wof a perfluorinated compound of formula (I)CF₃O—[CF(CF₃)CF₂O]_(n)—[CF₂O]_(m)—CF₃  (I) where n and m areindependently selected from 4 to 150, and further comprising an amountof hexamethylene tetramine (HMT) or analogue or derivative equivalent tothat contained within 0.5-10% w/w of a saturated aqueous solution ofHMT, saturation being determined at room temperature.
 2. A barrier creamcomposition comprising 30 to 100% w/w of a perfluorinated polymericcompound of formula (I) CF₃O—[CF(CF₃)CF₂O]_(n)—[CF₂O]_(m)—CF₃  (I) wheren and m are independently selected from 4 to 150, and further comprisingan HMT derivative of formula (II)

where R¹ is an organic group and X⁻ is an anion.
 3. A barrier creamcomposition according to claim 2, wherein R¹ is an optionallysubstituted straight or branched hydrocarbon chain having from 1 to 50carbon atoms.
 4. A barrier cream composition according to claim 2,wherein R¹ is an optionally substituted straight or branched hydrocarbonchain having from 1 to 50 carbon atoms and wherein the hydrocarbonchains are optionally substituted by one or more substituents selectedfrom aryl optionally substituted by halogen, and halogen.
 5. A barriercream composition according to claim 2, wherein R¹ is an optionallysubstituted straight or branched hydrocarbon chain having from 1 to 50carbon atoms and wherein the hydrocarbon chains are optionallysubstituted by one or more substituents selected from aryl optionallysubstituted by halogen, and halogen, and wherein R¹ is a fluorobenzyl ora group (CH₂)_(n)CH₃ where n is from 8 to
 20. 6. A process forprotecting the skin of a human or animal against volatile chemicalagents which consists of applying to the skin a barrier creamcomposition according to claim 1 or
 2. 7. A process for protecting theskin of a human or animal against volatile chemical agents whichconsists of applying to the skin a barrier cream composition accordingto claim 1 or 2.